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Involvement of Hyaluronan in the Adaptive Changes of the Rat Small www.nature.com/scientificreports OPEN Involvement of hyaluronan in the adaptive changes of the rat small intestine neuromuscular function after ischemia/reperfusion injury Michela Bistoletti1,4, Annalisa Bosi1,4, Ilaria Caon1,4, Anna Maria Chiaravalli2, Paola Moretto1, Angelo Genoni1, Elisabetta Moro3, Evgenia Karousou1, Manuela Viola1, Francesca Crema3, Andreina Baj1, Alberto Passi1, Davide Vigetti1,5* & Cristina Giaroni1,5* Intestinal ischemia/reperfusion (I/R) injury has severe consequences on myenteric neurons, which can be irreversibly compromised resulting in slowing of transit and hindered food digestion. Myenteric neurons synthesize hyaluronan (HA) to form a well-structured perineuronal net, which undergoes derangement when myenteric ganglia homeostasis is perturbed, i.e. during infammation. In this study we evaluated HA involvement in rat small intestine myenteric plexus after in vivo I/R injury induced by clamping a branch of the superior mesenteric artery for 60 min, followed by 24 h of reperfusion. In some experiments, 4-methylumbelliferone (4-MU, 25 mg/kg), a HA synthesis inhibitor, was intraperitoneally administered to normal (CTR), sham-operated (SH) and I/R animals for 24 h. In longitudinal muscle myenteric plexus (LMMP) whole-mount preparations, HA binding protein staining as well as HA levels were signifcantly higher in the I/R group, and were reduced after 4-MU treatment. HA synthase 1 and 2 (HAS1 and HAS2) labelled myenteric neurons and mRNA levels in LMMPs increased in the I/R group with respect to CTR, and were reduced by 4-MU. The efciency of the gastrointestinal transit was signifcantly reduced in I/R and 4-MU-treated I/R groups with respect to CTR and SH groups. In the 4-MU-treated I/R group gastric emptying was reduced with respect to the CTR, SH and I/R groups. Carbachol (CCh) and electrical feld (EFS, 0.1–40 Hz) stimulated contractions and EFS-induced (10 Hz) NANC relaxations were reduced in the I/R group with respect to both CTR and SH groups. After I/R, 4-MU treatment increased EFS contractions towards control values, but did not afect CCh-induced contractions. NANC on-relaxations after I/R were not infuenced by 4-MU treatment. Main alterations in the neurochemical coding of both excitatory (tachykinergic) and inhibitory pathways (iNOS, VIPergic) were also observed after I/R, and were infuenced by 4-MU administration. Overall, our data suggest that, after an intestinal I/R damage, changes of HA homeostasis in specifc myenteric neuron populations may infuence the efciency of the gastrointestinal transit. We cannot exclude that modulation of HA synthesis in these conditions may ameliorate derangement of the enteric motor function preventing, at least in part, the development of dysmotility. Te gut is one of the most sensitive organs to ischemia–reperfusion (I/R) injury, which may occur in multiple clinical conditions, such as sepsis, shock, trauma, surgery, and is associated with high morbidity and mortality1. 1Department of Medicine and Surgery, University of Insubria, via H. Dunant 5, Varese, Italy. 2Department of Pathology, ASST-Sette Laghi, Ospedale di Circolo Viale L. Borri 57, 21100 Varese, Italy. 3Department of Internal Medicine and Therapeutics, Section of Pharmacology, University of Pavia, Pavia, Italy. 4These authors contributed equally: Michela Bistoletti, Annalisa Bosi and Ilaria Caon. 5These authors jointly supervised this work: Davide Vigetti and Cristina Giaroni. *email: [email protected]; [email protected] SCIENTIFIC REPORTS | (2020) 10:11521 | https://doi.org/10.1038/s41598-020-67876-9 1 Vol.:(0123456789) www.nature.com/scientificreports/ Te initial ischemic insult causes mucosal shedding, barrier dysfunction and bacterial translocation with pro- longed reduction of intestinal blood fow1,2. Te successive reperfusion period, although essential to rescue ischemic tissues, paradoxically initiates a cascade of events that may lead to additional cell injury, exacerbating vascular and tissue damage3,4. Te I/R insult has severe consequences, not only on the metabolically active intestinal mucosa, but also on other cell types in the enteric microenvironment, including smooth muscle cells, enteric glial cells and neurons. Myenteric neurons are particularly sensitive and can be irreversibly compromised5. Te consequent neuronal loss may result in slowing of transit and hampered food digestion, suggestive of a long lasting neuropathy6,7. Te exact mechanisms involved in myenteric plexus alteration afer an intestinal I/R injury are largely unknown, although increased infammatory cell infltration, pro-infammatory cytokine release, production of reactive oxygen species (ROS) and increased levels of nitric oxide (NO) are certainly involved5,8,9. We have recently demonstrated that hyaluronan, HA, an unbranched glycosaminoglycan (GAG) component of the extracellular matrix (ECM), may provide an important perineuronal framework within myenteric ganglia, sustaining myenteric neuron homeostasis, similarly to the perineuronal net (PNN) found in the central nerv- ous system (CNS)10–12. Te PNN is an integral part of some neuronal populations within the CNS and regulates plasticity by controlling communication between neurons, synaptic ion sorting, lateral mobility of protein on the neuronal surface, and protects neurons from oxidative stress and neurotoxins12,13. Disruption of the PNN by matrix degrading enzymes is exacerbated by neuroinfammatory conditions and has been suggested to underlay development of neurological and neurodevelopmental disorders14. Analogously, during an experimental colitis in rats, in spite of the enhanced HA deposition in the whole intestinal wall, the well-organized HA network was lost, sustaining infammation-induced myenteric neuron damage 11. Accordingly, we hypothesize that HA may be mobilized also during an I/R injury in the gut, afecting myenteric neuron function. HA is synthesized by a family of three transmembrane synthases (HAS1, HAS2, HAS3) on the surface of neuronal plasma membrane, with diferent molecular weights and at diferent rates depending on the CNS area and developmental time points12,15. HASes are also localized inside the cell (nascent HASes are in the secretory pathway to reach the membrane or HASes are recycled from the membrane)16,17. Intriguingly, it is not fully understood whether intracellular HASes are active or not 18. Te biological efects of HA are stringently dependent upon the GAG size, the activation of downstream receptor pathways, and on its chemical modifcation driven by TNF-stimulated gene-6 (TSG6)19,20. Polymers of HA with high molecular weight have anti-infammatory properties, by recruiting diferent receptors, such as CD44, toll-like receptors (TLR) 4 and 2 and receptor for HA-mediated motility (RHAMM), towards cell membranes 15,21,22. However, during pathological states, including chronic infammation, long HA polymers are cut by hyaluronidases or oxidative stress by ROS into small frag- ments, which promote immune cell activation and production of pro-infammatory cytokines, thus favouring an increased infammatory response15,23. In a mouse model of colitis, for example, administration of medium molecular weight exogenous HA was efcacious in relieving infammation, via TLR4 activation 24,25, while accu- mulation of small endogenous HA fragments within the submucosal layer correlated with increased cytokine production, following leukocyte recruitment26,27. Seemingly, in the acute stage of stroke injury in man, increased production of low-molecular weight HA in leukocytes in the stroke and peri-infarcted region sustained the asso- ciated infammatory response, while activation of HA-induced cellular signalling in microvessels and neurons had a favourable impact on remodelling processes stimulating angiogenesis, revascularization and survival of susceptible neurons28. In this study, we focused on the impact of HA homeostasis changes in the adaptive responses of myenteric ganglia during an I/R injury in adult rats. Tis hypothesis was investigated with molecular, morphological and functional approaches. Some experiments were carried out afer in vivo treatment with 4-methylumbelliferone (4-MU), which reduces HA production by both depletion of cellular UDP-glucuronic acid necessary for HA synthesis and downregulation of HA synthases 29. Results General observations-histological assessment. Afer occlusion of the terminal branch of the superior mesenteric artery, the corresponding small intestinal segment became purple and returned to a normal pink color afer blood fow restoration. Animal recovered uneventfully from anesthesia and once awake were active, ate normally and did not show any sign of distress. A gross visual inspection of the regions subjected to I/R did not reveal major abnormalities with respect to sham-operated and un-operated control animals. Administra- tion of 4-MU, 25 mg/kg, did not induce signifcant gross morphology changes in intestinal segments obtained from animals subjected to I/R injury, sham-operated and controls, versus the respective untreated groups. In all experimental groups intestinal segments and mesenteric attachments were loser afer 4-MU treatment, com- pared to the untreated groups. In contrast with other studies, showing the development of mucosal lesions afer a transitory occlusion of the superior mesenteric artery in rats for 75 min followed by 24 h of reperfusion 8,30, in this study, the micro- scopic evaluation of intestinal sections afer I/R did not evidence prominent histological
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